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- 详细信息
- 文献和实验
- 技术资料
- 库存:
34
- 英文名:
Recombinant Human Androgen Receptor
- 保质期:
说明书
- 供应商:
上海抚生
- 保存条件:
-20°C for 12 months as lyophilized; 2-8°C for 1 month under sterile conditions after reconstitution
- 规格:
50μg/200μg/1mg
| 规格: | 50μg | 产品价格: | ¥3870.0 |
|---|---|---|---|
| 规格: | 200μg | 产品价格: | ¥7200.0 |
| 规格: | 1mg | 产品价格: | ¥22680.0 |
商品详情:
| 产品名称 | Recombinant Human Androgen Receptor |
| 货号 | A-01K100412 |
| 规格 | 50μg, 200μg, 1mg |
| 种属 | Human |
| 宿主 | E.Coli |
| 表达区间 | 650-920 |
| 分子量 | 29.7 kDa |
| 标签 | N-terminal His Tag |
| 纯度 | Greater than 95% by SDS-PAGE gel analyses |
| 应用 | Western Blot, ELISA |
| 性状 | Lyophilized from a 0.2 μm filtered solution in 10 mM Hepes, 500 mM NaCl with 5% trehalose, pH7.4 |
| 溶解方法 | Centrifuge the vial at 10,000 rpm for 1 minute, reconstitute at 200 μg/ml in sterile distilled water by gentle pipetting 2-3 times, don’t vortex |
| 存储 | -20°C for 12 months as lyophilized; 2-8°C for 1 month under sterile conditions after reconstitution |
| 别名 | AIS, ANDR_HUMAN, Androgen nuclear receptor variant 2, Androgen receptor, Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease), androgen receptor splice variant 4b, AR, AR8, DHTR, Dihydro testosterone receptor |
1. 分装量的多少取决于每次试验的使用量。分装量不少于 10μl;分装量越少,由于蒸发和吸附于管内壁而引起的损失会越大。
2. 避免使用无霜冰箱来保存重组蛋白。
3. 解冻后的分装重组蛋白应保存于 4℃,一般可以保存1-2周。
4. 稀释至工作浓度的重组蛋白最好不要在 4℃保存超过一天时间。
5. 冷冻保存的蛋白溶液,在使用之前应室温解冻,确认充分溶解,没有结块后使用。
6. 取用溶液时避免反复多次吹打蛋白溶液,以免引起不必要的活性损失。
操作步骤:
(一)获得目的基因
1、通过PCR方法:以含目的基因的克隆质粒为模板,按基因序列设计一对引物(在上游和下游引物分别引入不同的酶切位点),PCR循环获得所需基因片段。
2、通过RT-PCR方法:用TRIzol法从细胞或组织中提取总RNA,以mRNA为模板,逆转录形成cDNA第一链,以逆转录产物为模板进行PCR循环获得产物。
3、人工合成法:依照检索到的目的基因序列,设计合成相互重叠的单链寡核苷酸,再通过重叠延伸PCR法拼接出全长,这种方法无需模板,是目前常用的获取基因的手段之一。
(二)构建重组表达载体
1、载体酶切:将表达质粒用限制性内切酶(同引物的酶切位点)进行双酶切,酶切产物行琼脂糖电泳后,用胶回收Kit或冻融法回收载体大片段。
2、PCR产物双酶切后回收,在T4DNA连接酶作用下连接入载体。
(三)获得含重组表达质粒的表达菌种
1、将连接产物转化大肠杆菌,根据重组载体的标志(抗Amp或蓝白斑)作筛选,挑取单斑,碱裂解法小量抽提质粒,双酶切初步鉴定。
2、测序验证目的基因的插入方向及阅读框架均正确,进入下步操作。否则应筛选更多克隆,重复亚克隆或亚克隆至不同酶切位点。
3、以此重组质粒DNA转化表达宿主菌的感受态细胞。
(四)诱导表达
1、挑取含重组质粒的菌体单斑至2ml LB(含Amp50μg/ml)中37℃过夜培养。
2、按1∶50比例稀释过夜菌,一般将1ml菌加入到含50mlLB培养基的300ml培养瓶中,37℃震荡培养至OD600 ≌0.4-1.0(最好0.6,大约需3hr)。
3、取部分液体作为未诱导的对照组,余下的加入IPTG诱导剂至终浓度0.4mM作为实验组,两组继续37℃震荡培养3hr。
4、分别取菌体1ml,离心12000g×30s收获沉淀,用100μl 1%SDS重悬,混匀,70℃10min。
5、离心12000g×1min,取上清作为样品,可做SDS-PAGE等分析。
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. Purification of full-length androgen receptor (AR) is complicated by its low abundance, instability in the absence of androgen, and size and charge similarities with other nuclear proteins. Previous approaches to steroid hormone receptor purification
Analysis of Androgen Receptor SUMOylation
Androgen receptor (AR) is a ligand-controlled transcription factor that is deregulated and therefore targeted in prostate cancer. In addition to androgens, AR is regulated by post-translational modifications (PTMs). SUMOylation, conjugation
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